RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides withSTMN-2cryptic splicing and precedes clinical manifestation in ALS

Abstract

AbstractTDP-43 is an aggregation-prone protein which accumulates in the hallmark pathological inclusions of amyotrophic lateral sclerosis (ALS). However, analysis of deeply-phenotyped humanpost-mortemsamples has shown that TDP-43 aggregation, revealed by standard antibody methods, correlates poorly with symptom manifestation. Recent identification of cryptic-splicing events, such as the detection ofStathmin-2(STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism, but the temporal nature of TDP-43 loss and its relation to the disease process and clinical phenotype is not known. To address these outstanding questions, we used a novel RNA aptamer, TDP-43APT, to detect TDP-43 aggregation and used single moleculein situhybridization to sensitively reveal TDP-43 loss-of-function and applied these in a deeply-phenotyped humanpost-mortemtissue cohort. We demonstrate that TDP-43APTidentifies pathological TDP-43, detecting aggregation events that cannot be detected by classical antibody stains. We show that nuclear TDP-43 pathology is an early event, occurring prior to cytoplasmic aggregation and is associated with loss-of-function measured by coincidentSTMN-2cryptic splicing pathology. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function in the form of extensive cytoplasmic aggregation, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics as the presence ofSTMN-2cryptic exons and early TDP-43 aggregation events could be detected prior to symptom onset, holding promise for early intervention in ALS.Short AbstractRecent identification of cryptic-splicing events such as the detection ofStathmin-2(STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism in amyotrophic lateral sclerosis (ALS). However, the temporal nature of TDP-43 loss and its relation to clinical phenotype is not known. Here, we used a novel RNA aptamer to detect TDP-43 aggregation and used single molecule ISH to sensitively reveal TDP-43 loss-of-function, applying these methods in a deeply-phenotyped humanpost-mortemtissue cohort. We show that nuclear TDP-43 pathology is an early event, that coincides withSTMN-2cryptic splicing. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics and intervention prior to symptom onset in ALS.Graphical Abstract